Anti-HIV ribozymes

ABSTRACT

Ribozymes targeted against two portions of the HIV-1 genome were designed to cleave HIV RNA in the tat gene (TAT) or in a common exon for tat and rev (TR). The ribozymes were cloned into the LN (LTR-neomycin) retroviral vector plasmids and expressed as part of viral LTR-driven transcripts. The vectors were packaged as amphitropic virions and used to transduce human T-lymphocytes. Expression of the vector transcripts containing the ribozyme sequences were readily detected by Northern blot analysis of the transduced T cells. The T-lymphocytes expressing the anti-HIV-1 ribozymes showed resistance to HIV-1 replication.

This invention was made with government support under Grant No. J.R.NIAID R01 AI29329 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

This is a continuation of application Ser. No. 08/654,773, filed May 29,1996, now U.S. Pat. No. 5,695,938, which is a continuation ofapplication Ser. No. 08/355,244, filed Dec. 9, 1994, abandoned.

FIELD OF THE INVENTION

This invention relates to combinations of ribozymes. More particularly,the invention relates to combinations of two or more ribozymes, eachsuch ribozyme being targeted to a different portion of a viral genome,in particular an HIV-1 or other lentiviral genome.

ABBREVIATIONS

The following abbreviations are used in this application:

A=absorbance (1 cm)

AIDS=acquired immune deficiency syndrome

bp=base pair(s)

CMV=cytomegalovirus

DCT=double-copy tRNA

ELISA=enzyme-linked immunosorbent assay

HIV=human immuno-deficiency virus

IE=immediate early

kb=kilobase(s) or 1000 bp

LN=LTR-Nm

LTR=long terminal repeat

MoMuLV=Moloney murine leukemia virus

Nm=neomycin

nt=nucleotide(s)

rev (Rev)=regulates envelope expression

Rz=ribozyme(s)

tat (Tat)=transactivator of transcription

TR=common exon for tat and rev

BACKGROUND OF THE INVENTION

The concepts of genetic therapies for the treatment of genetic defectsor providing intracellular immunity to viral infection have beenentertained for a number of years (see Baltimore, 1988 (1); Szybalski,1992 (2)). Gene therapy has recently received more attention for itspotential utility in the treatment of HIV infection (Sarver and Rossi,1993 (3)). A number of different inhibitory strategies have been testedfor conferring resistance to HIV-1, including those encoding antisenseRNA, ribozymes (Rz), TAR or RRE decoys, trans-dominant mutant HIV-1genes and conditionally lethal toxins (reviewed in Sarver and Rossi,1993 (3)).

RNA-based strategies, such as antisense or Rz, have the dual advantagesof being sequence-specific, theoretically eliminating unwantedtoxicities, as well as not producing potentially immunogenic proteins. Asingle Rz molecule is capable of irreversibly inactivating multipletarget RNA molecules by sequential cycles of binding, cleavage andrelease, but even in the absence of multiple substrate turnover, Rzfunctionally inactivate target RNAs via cleavage (Zaug and Cech, 1986(4); Uhlenbeck, 1987 (5); Castanotto, et al., 1992 (6)).

Retroviral vectors currently comprise a relatively efficient system forgene transduction of mammalian cells, including human lymphocytes andhematopoietic cells (Mulligan, 1993 (7); Williams, 1990 (8)). Retroviralvectors and packaging cell lines have been developed which haveextremely low probabilities of producing replication-competentretroviruses and are increasingly used for clinical gene marking andgene therapy trials (Miller and Rosman, 1989 (9); Miller, 1990 (10);Anserson, et al., 1993 (11).

SUMMARY OF THE INVENTION

This invention provides two hammerhead Rz which are complementary to theHIV-1 genome, in the tat gene and a common exon of the tat and rev geneswhich are used in alternate reading frames. Genes encoding these Rz werecloned into the LTR-neo (LN) retroviral vector under the transcriptionalcontrol of two different RNA polymerase II and one RNA polymerase IIIpromoters. The vectors were packaged as amphitropic virions and used tointroduce the Rz into human T-lymphocytes. Expression of vector-derivedtranscripts containing the Rz in the human T-lymphocytes were examinedby Northern gel analyses. The most effective Rz expression was derivedfrom the MoMuLV LTR, which resulted in long, multifunctional, virallength transcripts. Cells transduced with these LTR driven Rz vectorconstructs were subsequently challenged by inoculations with HIV-1. TheT-lymphocytes which expressed catalytically active Rz displayedresistance to HIV-1 replication, whereas cells transduced bycatalytically inactive versions of the Rz were not resistant.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts ribozymes, target sequences and vectors. In FIG. 1A, theRz were designed to contain the consensus core hammerhead Rz catalyticdomain flanked by targeting sequences complementary to portions of theHXB2 molecular clone of the HIV-1 genome (Hahn, et al., 1984 (12)).Sequences and locations of the targeting sites of the anti-tat andtat-rev Rz in the HIV-1 genome are nt 5878 and 6025, respectively, inthe HIV IIIB isolate. These are schematically depicted as are the Rzbase pairing to their respective target RNAs. The (G) represents theposition of the nt change in the catalytically inactive mutant Rz. Sitesof RZ-mediated cleavage in the target sequences are indicated witharrows.

FIG. 1B depicts cleavage of tat and tat-rev RNAs in vitro. Equimolarmixtures of target RNAs and Rz were incubated at the specifiedtemperature for two hours. Lanes A, F, without Rz; lanes A, B, F, Gwithout 20 mM MgCl₂ ; lanes A through C, F, G, H, K, L., the cleavagereaction was at 37° C.; lanes D, I, M, the cleavage reaction was at 45°C.; lanes E, J, N, the cleavage reaction was at 55° C. P₁ (117 nt) andP₂ (78 nt) are cleavage products of the S₁ (195 nt) target of the TATRz, and P₃ (265 nt) and P₄ (152 nt) are cleavage products from the S₂(195 nt) TR Rz target. In lanes M and N, two new products can beobserved, one of which migrates just above P₃, while the otherco-migrates as a doublet with P₄. These represent products produced fromthe S₂ transcript as a consequence of cleavage by both the TAT and TRRz.

Methods. For in vitro RZ assays, each of the Rz was cloned into theNotI-SalI sites of the pBluescript vector SK(+) (Stratagene). Rz RNAswere prepared by transcription of SalI-linearized templates. The HIVsubstrates were prepared from a cloned SalI-SspI fragment of HIVHXB2 DNAcloned in pBluescript (+). The substrates S₁ and S₂ were transcribedfrom DdeI or AvaII digested templates, respectively.

FIG. 1C is a schematic representation of the Rz-expressing vectors. TheRz (R) were inserted into the BclI site, 31 bp upstream from the neogene start codon. Vectors contain the anti-tat Rz (L-TAT-neo), theanti-tat-rev Rz (L-TR-neo), both Rz in tandem (L-TR-TAT-neo) or the twomutant Rz (L-M-TR-TAT-neo) (see section a and panel A). The retroviralvector plasmid pLN was generously provided by A. D. Miller (FredHutchinson Cancer Center, Seattle, Wash., USA) and used for constructionof all the vectors (Miller and Rosman, 1989 (9)). The orientation andcopy number of the Rz in the vectors were confirmed by DNA sequencing.

Methods. PA317 amphitropic packaging cells (also provided by A. D.Miller) were grown in Dulbucceo's minimal essential media (DMEM), highglucose with 10% fetal calf serum (FCS) (Miller and Buttimore, 1986(13)). Human T-lymphocytes of the CEM cell line were obtained from theAmerican Type Culture Collection (Rockville, Md., USA) and maintained inRPMI with 10% FCS (R10 ). Clones of PA317 amphitropic packaging cellsproducing each retroviral vector at high titer were derived essentiallyas described with some modifications (Miller, et al., (13, 14). Inbrief, retroviral vector plasmid DNA was transfected into the PA317amphitropic packaging cell line using DOTAP transfection reagent(Boehringer-Mannheim, Indianapolis, Ind., USA). The transfected PA317cells were selected in 0.5 mg G418/ml (Geneticin, Bethesda ResearchLaboratories, Bethesda, Md., USA). Individual clones were expanded andscreened for the production of high titer of vector virus, which wasassessed by the transfer of G418 resistance to 3T3 cells. High titerclones were expanded and cryopreserved in multiple aliquots forsubsequent T lymphocyte transduction. A pre-established PA317 clonewhich produced the parental LN vector at high titer was obtained from A.D. Miller. T-lymphocytes were transduced by co-cultivation with the hightiter PA317 packaging cell clones. The PA317 vector-producing cells wereirradiated (4500 rad) and plated at a density of 2×10⁶ cells/100-mm dishin R10 medium. The next day, 1×10⁶ cells were added to each plate andthe medium was then supplemented with polybrene (Sigma, St. Louis, Mo.,USA) to a final concentration of 8 μg/ml. The T-lymphocytes and PA317cells were co-cultivated for 60 hours and then the non-adherent T-cellswere collected and cultured in R10 with 0.5 mgG418/ml until resistantcell pools were obtained (usually two to three weeks later).

FIG. 2 depicts Northern blot analysis of Rz expression in T-lymphocytes.Total cellular RNA was extracted from vector transduced T-cells andanalyzed by Northern blot. Total cellular RNA was extracted from thetransduced T-cells using the acid-phenol guanidinium thiocyanate method(Chomczynski and Sacchi, 1987 (15)). 20 μg of total cellular RNA weresubjected to electrophoresis in a 1% agarose gel containing 5.4%formaldehyde and transferred to nylon membranes. The blots were probedwith the Rz genes (as ds DNA removed from the vector plasmids), the neogene from LN, and the human β-actin cDNA (Gunning, et al., 1983 (16)),labeled with [α-³² P]dCTP by the random primer method (Feinberg andVogelstein, 1984 (17)). RNAs were from non-transduced T-lymphocytes(lane 1), and T-lymphocytes transduced by the LN vector (lane 2),L-TAT-neo (lane 3), L-TR-neo (lane 4), L-TR-tat-neo (lane 5) andL-M-TR-TAT-neo (lane 6). The same blot was successively hybridized withprobes consisting of a mixture of the TR and TAT Rz (top row), the neogene (middle row) and the human β-actin cDNA (bottom row).

FIG. 3 illustrates that the TAT and TR Rz expressed in T-cells inhibitHIV-1 replication. T-lymphocytes were challenged by infection with HIV-1on day 0. Samples of culture medium were collected and assayed for p24gag protein by ELISA at 2-3 day intervals. Cells studied were theparental T-lymphocytes (CEM, dotted squares), T-lymphocytes transduced bthe L-TR-neo vector (TR, closed diamonds), T-lymphocytes transduced bythe L-TAT-neo vector (TAT, thick squares), three separate pools ofT-lymphocytes transduced by the L-TR-TAT-neo vector (TR-TAT-1, 2 and 3,open diamonds, closed squares and open squares), or two separate poolsof T-cells transduced by the mutant Rz vector L-M-TR-TAT-neo(M-TR-TAT-1, 2, closed triangles and open triangles).

Methods. HIV-1 of the HTLV-IIIb strain was obtained from the AIDSResearch and Reference Reagent Program as a cell free concentrated virusstock at 2.91×10⁷ virus particles/ml. All work with HIV-1 was performedunder BL2 containment conditions. T-lymphocytes which had beentransduced by the retroviral vectors and selected in LG418 were assayedfor resistance to HIV infection. The Transduce dT-lymphocytes wereallowed to grow for at least one week in the absence of G418, prior toHIV-1 challenge. 1×10⁶ T-lymphocytes were pre-treated with polybrene (8μg/ml) in 2 ml of R10 for two hours at 37° C. The cells were then washedwith 10 ml of R10 and resuspended in 100 μl of HIV-1 diluted in R10(total of 150 TCID₅₀ per sample), and incubated for two hours at 37° C.After exposure to HIV-1, the cells were washed once with 10 ml of R10and 5×10⁵ cells were plated in 25 ml of R10 in flasks and incubated at37° C. Aliquots of medium were removed from the cultures at 2-3 dayintervals after infection and assayed for HIV replication bymeasurements of HIV-1 p24 antigen production, using an ELISA assay kit(Coulter, Hialeah, Fla., USA).

GENERAL DESCRIPTION AND EXEMPLIFICATION OF THE INVENTION

(a) Design of the Rz and retroviral vectors

Rz were designed to cleave sequences within the HIV-1 tat (TAT) gene anda tat/rev common exon (TR), as indicated in FIG. 1A. These sites arehighly conserved among all known HIV-1 isolates and present in all ofthe various unspliced, singly-spliced and multiply-spliced HIV-1 RNApresent in infected cells, as well as in the genomic RNA of the virion.Cleavage of the HIV-1 RNA at either of these sites would be expected toprevent translation of HIV-1 mRNA or reverse transcription of thegenome. In contrast, Rz targeted against structural genes, such as gag,pol or env, would not interfere with expression of the tat, rev or nef,because the target sequences within the structural genes are absent fromthe doubly spliced RNAs which encode the regulatory proteins.

To control for antisense effects of the Rz, mutant versions of each Rz,designated as M-TAT and M-TR, which have a nt substitution at anessential base of the active site and lack cleavage activity (data notshown), were also synthesized (FIG. 1A). The mutants have similarantisense effects to the Rz, but lack catalytic cleavage activity.Therefore, greater inhibitory activity against HIV-1 produced by thewild-type Rz over that seen with the mutant Rz vectors would imply theimportance of the Rz catalytic cleavage activity for the anti-viraleffects. The activities of TAT and TR Rz were first assayed utilizing anin vitro cleavage assay against synthetic transcripts which contain thespecific HIV-1 target sequences (FIG. 1B). Cleavage by the individual orcombined Rz resulted in the appearance of cleavage products of theexpected size. The combined Rz had a somewhat synergistic effectresulting in greater than 90% cleavage of the target transcript underthese conditions.

(b) Retroviral vectors to transduce the Rz.

The retroviral vector LN was used as a Rz delivery vehicle. The TAT orRz were cloned under the transcriptional control of either the viralMoMuLV, the human CMV IE promoter (Bahner eta 1., 1993 (18)), or adouble copy tRNA ^(Met) (DCT) promoter (Sullenger, et al., 1990 (19)).The resultant vectors were stably transduced into the human T-lymphocytecell line CME. Rz RNA expression from each of these promoters wasassayed via Northern gel analyses. The levels of expression from boththe CMV and DCT promoters were very weak in comparison to the LTR-driventranscripts (data not shown), and they were therefore excluded fromfurther use in these studies.

Either the individual TAT and TR Rz or a tandem combination of both Rz,or a combination of mutant versions of the two Rz, were cloned into theLN retroviral vector plasmid. Rz were inserted into the BclI site ofpLN, 31 bp upstream from the ATG start codon of the neo gene (FIG. 1C).These vectors would produce a single primary transcript of approximately2700 nt in length, from the transcriptional start point in the 5' LTRextending to the polyadenylation signal in the 3' LTR. The vectortranscript serves multiple functions including: (i) acting as the viralgenome of the amphitropic virion produced by the packaging cell line;(ii) encoding the neomycin phosphotransferase protein; and (iii)containing the Rz sequences. The presence of the 5' MoMuLV splice donorand a cryptic splice acceptor, located upstream from the BclI site intowhich the Rz were inserted, results in the production of a second,slightly shorter transcript, which would lack the packaging signal, butwould also contain the Rz and neo sequences. The Rz produced from thisvector would be flanked by approximately 1000 nt 5' and 1700 nt 3' ofthe Rz in a polyadenylated RNA.

(c) Expression of Rz in T lymphocytes

The Rz retroviral vectors were packaged as amphitropic virions in thePA317 cell line and used to transduce human T-lymphocytes. Followinggene transfer, the cells were placed under selection with the Nmanalogue G418 to produce pools of cells which contain the vectors.Control cells were produced by transduction with the parental neo genevector, LN, which lacks Rz sequences, followed by selection in G418.

Expression of RNA transcripts by the different vector constructs in theT-lymphocytes was analyzed by Northern blot analyses. As expected andillustrated in FIG. 2, the parental T-cells and T-cells transduced bythe parental LN vector did not show any RNA which hybridized with the Rzprobes (lanes 1 and 2). The full-length vector transcripts from the LTRwere seen at the expected size of 2700 nt in the T-lymphocytestransduced by each of the Rz vectors. The levels of the vector-specifictranscripts were very similar in each of the cell pools. Thesingly-spliced vector transcripts were not seen in this exposure, butfollowing a prolonged exposure of the blot (twenty days), fainthybridization bands of the predicted sizes were seen, suggesting thatmost of the vector transcripts remained unspliced in the T-cells.Re-probing of the blot with the neo gene showed the same vectortranscripts as seen with the Rz probe and also demonstrated the presenceof a similar transcript in the LN transduced cells, which lacked the Rzsequences. Re-probing a third time with a human β-actin cDNA verifiedthat nearly equal amounts of RNA were analyzed from each cell pool (FIG.2).

(d) Inhibition of HIV-1 replication in T-lymphocytes

To test the ability of the Rz to inhibit HIV-1 replication, theRz-transduced cells with infectious HIV-1 IIIb were challenged. Samplesof the culture supernatant were collected at various times and assayedby ELISA for the presence of HIV-1 p24 antigen. Three separate pools ofT-cells transduced by L-TR-TAT-neo (TR-TAT-1, 2 and 3) were prepared andanalyzed. Two sets of control cells were used: non-transducedT-lymphocytes and T-lymphocytes transduced with the parental LN vectorand subjected to G418 selection.

The results of an HIV-1 challenge experiment are shown in FIG. 3.Control T-cells readily supported HIV-1 replication, with the virusgrowing-out by twelve days after infection. Compared to control cells,T-cells transduced by either the single Rz L-TAT-neo or L-TR-neo ortandem Rz L-TR-TAT-neo showed resistance to HIV infection, with viralout-growth delayed to at least day 20. Nearly equal levels of inhibitionwere seen with vectors carrying either the TAT or TR Rz or with thevector containing the two Rz in tandem.

The results of this experiment (experiment 1) and a second separateassay (experiment 2) are summarized in Table I. In the secondexperiment, HIV-1 out-growth from the control cells occurred by day 9.The cells transduced by the Rz vectors again showed inhibition of HIV-1growth, with virus production delayed until days 12-15.

                  TABLE I                                                         ______________________________________                                        Effects of ribozymes on HIV-1                                                   replication in T-lymphocytes                                                               Day of HIV-1 out-growth*                                       Vector         Experiment 1                                                                             Experiment 2                                        ______________________________________                                        None           12         9                                                     LN n.d. 9                                                                     L-TAT-neo  20 15                                                              L-TR-neo  >20 12                                                              L-TR-TAT-neo                                                                  Pool 1 20 15                                                                  Pool 2 >20 15                                                                 Pool 3 >20 15                                                                 L-M-TR-TAT-neo                                                                Pool 1 12 12                                                                  Pool 3 15 12                                                                ______________________________________                                         *Out-growth was determined by an ELISA measurement of p24 protein levels      exceeding an A.sub.420 nm of 0.20.                                            n.d., not done.                                                          

As a control for inhibition of HIV-1 due to antisense effects of thesequences flanking the catalytic domain which are complementary toHIV-1, a vector was made containing mutant versions of the TR/TAT Rzdimer. The mutants contain substitutions of one base within each of thekey catalytic hammerhead regions, which has been observed to completelyeliminate in vitro cleavage activity. The T-lymphocytes with the mutanttandem Rz (L-M-lTR-lTAT-neo) showed little or no inhibition of HIV-1replications. These findings are consistent with the essential role ofthe catalytic Rz domain in cleaving viral RNA to produce inhibition ofHIV-1 growth.

(e) Conclusions

1. Regulatory gene encoding transcripts as Rz or antisense targets

The earliest RNA transcripts from HIV-1 which appear in the cytoplasmare doubly spliced and encode the regulatory genes tat, rev and nef,these transcripts contain the target sequences for the anti-tat (TAT)and tat-rev (TR) Rz. Thus, inhibition of expression by these Rz would beexpected for the genes of the regulatory proteins tat, rev and nef, aswell as for those encoding the virion structural and enzymatic proteins.A vector containing both TAT and TR Rz in tandem (L-TR-TAT-neo) wasequally inhibitory to the vectors which contained either of the Rzsingly (L-TAT-neo or L-TR-neo). Although the tandem Rz vector was notfound to be superior to the single Rz vectors in the cell culture assay,there may be in vivo advantages to having multiple Rz against differentHIV-1 target sequences in a single vector. Potentially, multimeric Rztargeting different portions of the genome may be less susceptible tothe loss of inhibition, due to the development of sequence heterogeneityby HIV-1 at the target site, than would be a single Rz (Chen, et al.,1992 (20)).

The results comparing functional and mutant, inactive Rz document theimportance of a functional catalytic Rz core for the inhibition ofHIV-1. The results are somewhat in contradiction to those of Lo et al.(1992) (21), who found that a retroviral vector expressing an antisenseRNA to a nearby site of the HIV-1 tat gene, but lacking any Rz catalyticdomain, was more inhibitory to HIV-1 than was a vector expressing ahammerhead Rz to the same site as the one which was targeted.

2. Improving Rz efficacy

The vector constructs which were used were relatively simple, containingonly a single transcriptional unit from the MoMuLV Ltr. Transcriptscontaining the Rz were readily detected by Northern blot analysis. Itwas found that this type of vector produced higher levels of Rz RNA thandid vectors in which the Rz was expressed from either an internal CMVpromoter or a pair of tRNA^(Met) promoters contained within the vectorLTR (C.Z. and D.B.K., data not shown). The steady-state levels of Rzwhich accumulate in cells is a function of both the rate of theirproduction as well as degradation. It may be possible to achieve moreinhibition of HIV-1 with higher intracellular levels of Rz by using moreactive transcriptional units or by adding sequences, such as stem loops,which further stabilize the Rz transcripts. In the L-RZ-neo type vector,the Rz are contained within long viral transcripts of almost 3000 nt.The ability of the Rz to have significant cleavage activity whichinhibits HIV-1 replication in the context of such long transcripts wassomewhat surprising. One possible explanation for this observedfunctional activity in this unlikely RNA context is that the HIV-1 andRz transcripts are co-localizing to the same intracellular sites. Thismay be a consequence of the relative lack of splicing of the Rztranscripts, forcing them into the same cellular pathway as the HIV-1full-length, unspliced viral transcripts. Co-localization of Rz andtarget RNAs for retroviral encoded transcripts has been demonstrated bySullenger and Cech (1993) (22), although in their example bothtranscripts shared the same MoMuLV packaging signals. Efficacy with thelong viral transcripts needs to be further examined to determine whetheror not this is a function of intracellular co-localization.

3. Rz as inhibitors of HIV replication

Sarver et al. (1993) (23) first reported intracellular cleavage of HIV-1RNA by a Rz. In their study, a hammerhead Rz directed against the 5'part of the gag gene expressed from a plasmid with the human β-actinpromoter was introduced by transfection into CD4+HeLa cells. The cellstransduced by the Rz showed decreased levels of HIV-1 RNA and p24release after infection by HIV-1. Weerasinghe, et al., (1991) (24)produced a hammerhead Rz directed against the 5' leader region of HIV-1in a variety of retroviral vector constructs and transduced the MT₄human T-cell line. HIV-1 replication was inhibited most effectively whenthe Rz was expressed under control of a fusion promoter consisting ofthe Herpes simplex thymidine kinase promoter and the HIV-1 TAR element.Dropulic et al., (1992) (25) demonstrated that a hammerhead Rz directedagainst the U5 region of the HIV-1 genome, expressed undertranscriptional control of the MoMuLV LTR of a retroviral vector,inhibited HIV-1 replication in chronically infected H9 T-lymphocytes andMT₄ cells. Chen, et al., (1992) (20) produced a multimeric complex ofnine Rz directed against different targets of the HIV-1 env region whichinhibited HIV-1 in a transient transfection assay. Yu, et al. (1993)(26) have reported that a hairpin Rz directed against the 5' leaderregion of HIV-1 inhibited HIV-1 expressed by transient transfectionassays in HeLa cells. The use of this hairpin Rz has recently beenapproved by the NIH DNA Recombinant Advisory Committee for a clinicaltrial for transduction of patients' peripheral blood T-lymphocytes.Thus, a variety of Rz expression vectors have shown promise asanti-HIV-1 agents.

4. Potential advantages of Rz as anti-viral therapeutic agents

Rz would be expected to lack immunogenicity because they do not encodeproteins, in contrast to strategies, such as trans-dominant inhibitorymutants, which rely on the production of foreign proteins. Thespecificity for inhibiting complementary sequences of nucleic acids mayminimize interference with normal cellular functions. RNA decoys whichcompete for either the TAT or REV proteins may also sequester normalcellular proteins which bind to the RNA domains directly or throughbinding to the HIV-1 proteins. Additional studies to identify optimaltranscriptional control elements to maximize Rz production with theoptimal intracellular localization must also be performed. Finally,clinical trials will be needed to assess whether peripheral bloodT-lymphocytes or bone marrow stem cells afford better targets for Rzgene therapy of AIDS.

BIBLIOGRAPHY

1. Baltimore, D., Nature 335: 395-396 (1988)

2. Szbalski, W., BioEssays 14: 495-500 (1992)

3. Sarver, N., et al., J.NIH Res. 5: 63-67 (1993)

4. Zaug, A. J., et al., Science 231: 470-475 (1986)

5. Uhlenbeck, O. C. Nature 328: 596-600 (1987)

6. Castanotto, D., et al., Crit. Rev. Euk. Gene Exp. 2: 331-357 (1992)

7. Mulligan, R. C., Science 260: 926-932 (1993)

8. Williams, D. A., Hum. Gene Ther. 1: 229-239 (1990)

9. Miller, A. D., Biotechniques 7: 980-990 (1989)

10. Miller, A. D., Hum. Gene Ther. 1: 5-14 (1990)

11. Anderson, W. F., et al., Hum. Gene Ther. 4: 311-321 (1993)

12. Hahn, B. H., et al., Nature 312: 166-169 (1984)

13. Miller, A. D., et al., Mol. Cell. Biol. 6: 2895-2902 (1986)

14. Miller, A. D., et al., Cell Mol. Genet. 12: 175-183 (1986)

15. Chomczynski, P., et al., Anal. Biochem. 162: 156-159 (1987)

16. Gunning, P., et al., Mol. Cell. Biol. 3: 787-795 (1983)

17. Feinberg, A. P., et al., Anal. Biochem 137: 266 (1984)

18. Bahner, I., et al., J. Virol. 67: 3199-3207 (1993)

19. Sullenger, B. A., et al., Mol. Cell. Biol. 10: 6512-6523 (1990)

20. Chen, C. J., et al., Nucleic Acids Res. 20: 4581-4589 (1992)

21. Lo, K., et al., Virology 190: 176-183 (1992)

22. Sullenger, B. A., et al., Science 262: 1566-1569 (1993)

23. Sarver, N., et al., J. NIH Res. 5: 63-67 (1993)

24. Weerasinghe, M., et al., J. Virol. 65: 5531-5534 (1991)

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26. Yu, M., et al., Proc. Natl. Acad. Sci. USA 90: 6340-6344 (1993)

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCAGGAAGUC AGCCUAAAA             - #                  - #                      - # 19                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41                                                                (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GGUCCUUCAA GAGCAGGAGU GCCUGAGUAG UCUCGGAUUU U    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CAGACUCAUC AAGCUUCUC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41                                                                (B) TYPE: nucleotide                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GUCUGAGUAA GAGCAGGAGU GCCUGAGUAG UCUUCGAAGA G    - #                      - #   41                                                                    __________________________________________________________________________

I claim:
 1. A human T-lymphocyte transduced in vitro with a vectorcomprising genes encoding first and second ribozyme moieties, said firstribozyme moiety being designed to cleave HIV-1 RNA in the tat gene at anucleotide within SEQ ID NO: 1 and said second ribozyme moiety beingdesigned to cleave HIV-1 RNA in a common exon for the tat and rev genesat a nucleotide within SEQ ID NO:
 3. 2. The human T-lymphocyte of claim1, wherein said vector is a retroviral vector and said genes encodingfirst and second ribozyme moieties are transcribed under the control ofa long terminal repeat sequence.
 3. An HIV-1 infected mammalian celltransduced in vitro with a viral vector comprising genes encoding aribozyme designed to cleave a sequence within the HIV-1 tat gene at anucleotide within SEQ ID NO: 1 and a ribozyme designed to cleave asequence within the HIV-1 tat/rev common exon at a nucleotide within SEQID NO:
 3. 4. A human T-lymphocyte transduced in vitro with a retroviralvector comprising genes encoding a ribozyme moiety designed to cleaveHIV-1 RNA in the tat gene at a nucleotide within SEQ ID NO: 1 and aribozyme moiety designed to cleave HIV-1 RNA in the tat/rev common exonat a nucleotide within SEQ ID NO: 3, wherein said genes encoding saidribozyme moieties are under the transcriptional control of a MoMuLVpromoter.
 5. The human T-lymphocyte of claim 4, wherein said vectorproduces a single viral transcript comprising the transcriptionalsequences of said ribozyme moieties.
 6. The human T-lymphocyte of claim1, wherein the first ribozyme moiety is designed to cleave HIV-1 RNA inthe tat gene at nucleotide 5878 and the second ribozyme moiety isdesigned to cleave HIV-1 RNA in a common exon for the tat and rev genesat nucleotide
 6025. 7. The human T-lymphocyte of claim 2, wherein thefirst ribozyme moiety is designed to cleave HIV-1 RNA in the tat gene atnucleotide 5878 and the second ribozyme moiety is designed to cleaveHIV-1 RNA in a common exon for the tat and rev genes at nucleotide 6025.8. The HIV-1 infected mammalian cell of claim 3, wherein the viralvector comprises genes encoding a ribozyme designed to cleave a sequencewithin the HIV-1 tat gene at nucleotide 5878 and a ribozyme designed tocleave a sequence within the HIV-1 tat/rev common exon at nucleotide6025.
 9. The human T-lymphocyte of claim 4, wherein the retroviralvector comprises genes encoding a ribozyme moiety designed to cleaveHIV-1 RNA in the tat gene at nucleotide 5878 and a ribozyme moietydesigned to cleave HIV-1 RNA in the tat/rev common exon at nucleotide6025.
 10. The human T-lymphocyte of claim 5, wherein the retroviralvector comprises genes encoding a ribozyme moiety designed to cleaveHIV-1 RNA in the tat gene at nucleotide 5878 and a ribozyme moietydesigned to cleave HIV-1 RNA in the tat/rev common exon at nucleotide6025.